Complications of atherosclerosis are the leading cause of mortality in developed countries. More than 100 genes that can influence atherogenesis have been identified. Our long-term goals are to elucidate the signaling dependent transcriptional mechanisms of these genes. Recently, we identified a novel transcriptional mechanism by which FOXO4 regulates smooth muscle cell (SMC) phenotypes and transcription of matrix metalloproteinase 9 (MMP9) and Toll-like-receptor 4 (TLR4). As SMC phenotypic modulation, MMP9, and TLR4 are involved in atherogenesis, we propose that FOXO4 contributes to atherosclerosis through these effectors. Three specific aims are proposed. (1) To characterize the signaling pathways that regulate FOXO4 activities. We will investigate whether FOXO4 mediates JNK-activated MMP9 transcription and whether JAK/STAT signaling suppresses MMP9 transcription through inactivation of FOXO4 by PIM kinases. (2) To investigate the role of FOXO4 in transcriptional activation of TLR4. We will study the molecular mechanism by which FOXO4 activates TLR transcription and examine whether upregulation of TLR4 by FOXO4 is sufficient to promote TLR4-mediated proinflammatory cytokine production. We will also investigate whether FOXO4 plays a role during monocyte to macrophage differentiation. (3) To determine whether and how FOXO4 promotes atherosclerosis. We will test whether inactivation of Foxo4 in apoE-null mice reduces atherosclerosis and whether Foxo4 influences atherosclerosis through downstream effectors (TLR4, MMP9), and/or SMC phenotypic modulation. The lack of response to lipid-lowering drugs in patients with cardiovascular disease emphasizes the need to identify new therapeutic targets for the treatment of atherosclerosis. FOXO4 is a promising target for therapeutic intervention since inhibition of FOXO4 activity may reduce TLR4-induced cytokine production, reduce intimal thickening, and stabilize late atherosclerotic plaques. Accomplishing the specific aims in this proposal will provide the foundation to assess this possibility. While our focus in this proposal is to understand the role of FOXO4 in atherosclerosis, studies of the specific aims 1 &2 will also help us to understand the role of FOXO4 in other human diseases such as rheumatoid arthritis, multiple sclerosis, and cancer, as MMP9 and/or TLR4 are implicated in the pathogenesis of these diseases as well.